A note on genetic analysis in Madras Red sheep (Ovis aries) of India using microsatellite markers

S Prema,  S N Sivaselvam and S M K Karthickeyan  

Department of Animal Genetics & Breeding, Madras Veterinary College, Tamilnadu Veterinary and Animal Sciences University, Chennai-600 007, India
snsivaselvam@hotmail.com

Abstract 

Molecular characterization of Madras Red sheep was done using 17 ovine-specific microsatellite markers. The number of observed alleles ranged from 3 to 8 with a mean of 5 across all loci.

The size of alleles ranged from 74 to 222 bp with an overall frequency of 0.0208 to 0.6250. In total, 85 alleles were observed at the 17 loci studied. All the 17 loci were found to be highly polymorphic. The indicators of genetic variations such as polymorphism information content and expected heterozygosity were found to be 0.6667 and 0.7168 respectively. The overall F_IS value (0.0213) indicated a considerable deficit of heterozygosity in Madras Red population.

Key words: Madras Red sheep, genetic structure, microsatellite markers

Introduction 

Madras Red sheep, widely distributed in six northern districts of Tamil Nadu state in India, is reared mainly for mutton production. The population of Madras Red sheep was 0.843 million as per the 17^th Livestock Census (2003). The sheep are medium-sized animals with predominant body colour being brown, the intensity of which varies from light tan to dark brown; some animals have white markings on the forehead, inside the thighs and on the lower abdomen. Characteristically, rams have strong corrugated and twisted horns while the ewes are polled (Acharya 1982). The first and foremost step for the sustainable use of such domestic animal genetic resources is the gathering of information about the genetic variability within and between breeds through characterization. Earlier, phenotypic traits (such as coat colour, horn types, wool characteristics) or cytogenetic markers or biochemical markers were used to characterize a breed. Owing to their limitations in identifying and resolving the differences between closely related breeds, the molecular characterization was felt necessary to understand the underlying genetic variation. Of several molecular marker systems such as RFLP, RAPD, AFLP and microsatellites, the microsatellites are highly polymorphic, abundant and distributed throughout the genome. They have large number of alleles, with a high level of heterozygosity, inherited in Mendelian fashion and are capable of detecting variations at molecular level. The present study was taken up in Madras Red sheep using microsatellite markers to find out the genetic structure.

Materials and methods 

A total of 50 blood samples were randomly collected from the breeding tract of Madras Red sheep which is very vast and wide covering about six districts of Tamilnadu state. The genomic DNA was isolated by a rapid non-enzymatic method described by Lahiri and Nurnberger (1991). A panel of 17 microsatellite markers (Table 1) selected from the list of markers suggested by the International Society of Animal Genetics (ISAG) under FAO’s programme (MoDAD) were used for the microsatellite analysis.

The amplification reactions were carried out using a programmable thermal cycler (MJ Research, USA) with the PCR reaction mixture of 20μl. The reaction mixture was prepared by adding 50-100 ng of template DNA; 5 picomoles each of forward and reverse primers; 100 mM dNTPs; 0.75 units of Taq DNA polymerase. The amplification was carried out with annealing temperature ranging from 50 to 63º C for different primers for 35 cycles. The amplified PCR products were verified on 2% agarose gel and denatured at 95º C for 5 minutes. The denatured samples were electrophoresed on a standard 6 per cent denaturing polyacrylamide gel at 65 W. The resolved bands were visualized after silver staining as described by Cominicini et al (1995).    

The allele sizes were scored using Diversity Database software (BioRad, USA). The POPGENE software, version 1.31 (http://www.ualberta.ca/~fyeh) was used to calculate the effective number of alleles, allele frequencies and heterozygosity. The polymorphism information content was calculated using the individual frequencies in which the alleles occur at each locus using Nei’s formula (1978). The expected genotypes were compared with observed genotypes in the test for goodness of fit to assess whether the population was in Hardy-Weinberg equilibrium or not.

Results and discussion 

All the 17 microsatellite loci amplified successfully and were found to be polymorphic. The parameters of genetic variability estimated have been furnished in Table 1.

Microsatellite alleles

The number of alleles observed across all loci ranged from 3 (BM6526) to 8 (OarCP38) with a mean of 4.94 ± 0.14. The size of alleles ranged from 74 (OarCP20) to 222 bp (BM827). The frequency of alleles ranged from 0.020 (CSSM47) to 0.480 (OarCP20). In Indian breeds of sheep, similar ranges of observed number of alleles between 2 and 8 with an average of 5.04 in Muzzafarnagri (Arora and Bhatia 2004) and from 3 to 10 in Nali and 2 to 8 in Chokla (Sodhi et al 2006) were reported. Whereas, higher average number of alleles of 6.2 in Garole sheep (Sodhi et al 2003) and 5.7 in Magra sheep (Arora and Bhatia 2006) were reported. Among the exotic sheep, Gutierrez-Espelata et al (2000) reported 2 to 4 number of alleles in Bighorn sheep and Wafula et al (2005) reported 6 to 7 alleles in West African Djallonke sheep. Saitbekova et al (2001) observed similar number of alleles for the loci OarCP20, OarFCB20 and OarFCB48 in Swiss sheep breeds. The effective number of alleles ranged from 2.9 (OarVH72) to 4.6 (OarHH41) with a mean of 3.7 which is similar to the reports of Sodhi et al (2003) in Garole sheep, Arora and Bhatia (2004) in Muzzafarnagri,   Sodhi et al (2006) in Nali and Chokla and  Arora and Bhatia (2006) in Magra sheep breeds.

Polymorphism information content

The polymorphism information content (PIC) values for all the 17 loci ranged from 0.454 (BM6506) to 0.785 (OarFCB48). In the present study, all the 17 microsatellite loci showed higher PIC values indicating suitability of these loci for genetic studies in sheep. Similarly higher PIC values had been reported in Laxta (Arranz et al 2001), Uruguayan (Tomasco et al 2002), Muzzafarnagri (Arora and Bhatia 2004), Pag Island (Ivankovic et al 2005), Nali and Chokla (Sodhi et al 2006) and Magra (Arora and Bhatia 2006) breeds of sheep.

Hardy-Weinberg equilibrium

The results of the test of goodness of fit are furnished in Table 1. The results revealed that the population was in Hardy-Weinberg equilibrium proportions at BM1314, BM6526, BM757, OarCP38, OarHH41 and OMHC1 loci. The disequilibria observed in the remaining loci might be due to both the systematic and dispersive processes operating in the population. Similarly, several authors (Diez-Tascon et al 2000, Tomasco et al 2002, Ivankovic et al 2005) reported the departure of the sheep populations from Hardy-Weinberg equilibrium at various loci.

Estimation of heterozygosity

Genetic variability can be measured as the amount of actual or potential heterozygosity. The observed heterozygosity ranged from 0.551 (OMHC1) to 0.776 (OarCP38) with a mean of 0.697 and the expected heterozygosity ranged from 0.554 (BM6506) to 0.796 (CSSM47) with a mean of 0.706. The high mean heterozygosity value indicates low level of inbreeding as a result of wider geographical distribution of the breed and the practice of periodical exchange of breeding rams among the flocks. The high heterozygosity values observed in Madras Red sheep were similar to those of sheep breeds investigated earlier (0.68 in French Mutton Merino by Diez-Tascon et al (2000); 0.67 in Swiss sheep breeds by Saitbekova et al (2001); 0.67 in Garole sheep by Sodhi et al (2003); 0.70 in Muzzafarnagri sheep by Arora and Bhatia (2004); 0.71 in Baltic sheep breeds by Tapio et al (2005); 0.83 in Pag Island sheep by Ivankovic et al (2005); 0.74 in Czech and Slovak sheep breeds by Jandurova et al (2005);  0.67 in Nali and Chokla sheep breeds by Sodhi et al (2006); 0.69 in Magra sheep by Arora and Bhatia (2006).

Within population inbreeding estimate

The within population inbreeding estimates (F_IS) calculated in the present study are presented in Table 1. The values ranged from -0.074 to 0.298 with a mean of 0.021.  Out of seventeen loci studied, eight revealed negative F_IS values indicating excess of heterozygotes in the population. Out of the remaining nine positive F_IS  values, six values were higher (ranging between 0.106 to 0.298) which had hiked the overall mean of the within population inbreeding estimate. The relatively higher values of F_IS in the present study may be due to heterozygote deficiencies within the selected loci in the population. However, the possibility of the Wahlund effect leading to these heterozygote deficiencies could not be ruled out. But a high rate of inbreeding was observed in Nali (0.397) and Chokla (0.299) sheep by Sodhi et al (2006), in Sarda sheep (0.19) by Pariset et al (2003), in Muzzafarnagari sheep (0.058) by Arora and Bhatia (2004) and in Magra sheep (0.16) by Arora and Bhatia (2006).

The present study proves that the microsatellites used are highly polymorphic and are informative for molecular characterization and it also could be used for exploitation of genetic variability of the population for conservation. Similar work has to be carried out for the remaining breeds of sheep in India to define the genetic relationship among them through evolutionary lineage.

Acknowledgements 

The authors are thankful to the Principal Investigator, ICAR-NBAGR Core Laboratory, Department of Animal Genetics and Breeding, Madras Veterinary College, Chennai for providing laboratory facilities.

References 

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Received 18 October 2007; Accepted 22 August 2008; Published 6 November 2008

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